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Characterisation of the enzymatic properties of MpAPr1, an aspartic protease secreted by the wine yeast Metschnikowia pulcherrima
Author(s) -
Theron Louwrens Wiid,
Bely Marina,
Divol Benoit
Publication year - 2017
Publication title -
journal of the science of food and agriculture
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.782
H-Index - 142
eISSN - 1097-0010
pISSN - 0022-5142
DOI - 10.1002/jsfa.8217
Subject(s) - pichia pastoris , protease , yeast , wine , biochemistry , enzyme , chemistry , winemaking , casein , heterologous expression , food science , enzyme assay , specific activity , chromatography , biology , recombinant dna , gene
BACKGROUND MpAPr1 , encoding an acid protease from the wine yeast Metschnikowia pulcherrima IWBT Y1123, was previously isolated and shown to display potential activity against casein and grape proteins. However, its characterisation remained partial. RESULTS MpAPr1 was cloned into the pGAPZαA vector and transformed into Komagataella pastoris X33 for heterologous expression. After verification of activity, the enzyme properties were characterised. Protease activity within the concentrated supernatant was retained over a pH range of 3.0 to 5.0 and between 10 °C and 50 °C. Optimal conditions for protease activity were found at 40 °C and pH 4.5. Activity was mostly unaffected by the presence of metal ions with the exception of Cu 2+ and Ni 2+ . Furthermore, proteolytic activity was retained in the presence of sugar and ethanol. pH and temperature conditions for MpAPr1 expression in K. pastoris were optimised. Purification was achieved by means of cation exchange chromatography and kinetic parameters ( K m and V max ) were determined. MpAPr1 activity against grape proteins was confirmed, but the extent of the degradation was dependent on the nature of these proteins and the environmental conditions. CONCLUSION Overall, the results suggest that MpAPr1 could be applied in food biotechnology processes such as winemaking. © 2017 Society of Chemical Industry